We describe, for the first time, the use of cellular plastic compressed collagen as a substrate for human limbal epithelial cell expansion and stratification. The characteristics of expanded limbal epithelial cells on either acellular collagen constructs or those containing human limbal fibroblasts were compared to a human central cornea control. After compression, human fibroblasts in collagen constructs remained viable and limbal epithelial cells were successfully expanded on the surface. After airlifting, a multilayered epithelium formed with epithelial cell morphology very similar to that of cells in the central cornea. Immunochemical staining revealed expression of basement membrane proteins and differentiated epithelial cell markers found in native central cornea. Ultrastructural analysis revealed cells on collagen constructs had many features similar to central cornea, including polygonal, tightly opposed surface epithelial cells with microvilli and numerous desmosomes at cell–cell junctions. Taken together, these data demonstrate that plastic compressed collagen constructs can form the basis of a biomimetic tissue model for in vitro testing and could potentially provide a suitable alternative to amniotic membrane as a substrate for limbal epithelial cell transplantation.